Protein sequencing method and reagents

Abstract

The invention describes methods and reagents useful for sequencing polypeptide molecules. The method comprises affixing a polypeptide to a substrate and contacting the polypeptide with a plurality of probes. Each probe selectively binds to an N-terminal amino acid or an N-terminal amino acid derivative. Probes bound to the polypeptide molecule are then identified before cleaving the N-terminal amino acid or N-terminal amino acid derivative of the polypeptide. Also provided are methods for the sequencing a plurality of polypeptide molecules in a sample and probes specific for N-terminal amino acids or N-terminal amino acid derivatives.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. provisional application no. 61 / 245,875 titled SINGLE MOLECULE PROTEIN SEQUENCING METHOD filed on Sep. 25, 2009, the contents which of are herein incorporated by reference.NON-PUBLICATION REQUEST[0002]A non-publication request has been submitted with this application upon filing. This application is not to be published under 35 U.S.C. 122(b)INCORPORATION OF SEQUENCE LISTING[0003]A computer readable form of the Sequence Listing “13795-15_Sequence_Listing.txt” (4,636 bytes), submitted via EFS-WEB and created on Sep. 27, 2010, is herein incorporated by reference.FIELD OF THE INVENTION[0004]This invention relates to the field of protein sequencing. More specifically, the invention relates to methods, assays and reagents for sequencing protein or polypeptide molecules as well as to methods and assays for the parallel sequencing of proteins or polypeptides.BACKGROUND OF THE INVENTION[0005]Proteins mediate...

AI Technical Summary

Problems solved by technology

The identification and quantification of proteins present in biological samples is therefore a fundamental problem applicable to most biomedical research studies, and a cornerstone of the emerging field of Proteomics.

These methods are generally low-throughput, computationally demanding and require the use of expensive equipment.

However, even the most sensitive mass spectrometers require relatively large amounts of sample, with current limits of detection on the order of 108 molecules (equivalent to nanogram or femtomole levels) and are not able to exhaustively sequence complex mixtures of proteins due to ion-ion interference, preferential (biased) detection of certain molecules, limited dynamic range and general under-sampling.

While dramatic improvements have been made in the past couple of years with respect to the speed, comprehensiveness and availability of high-throughput massively parallel DNA sequencing platforms capable of sequencing large numbers of different nucleic acid molecules simultaneously, advances in mass spectrometer performance have been incremental.

Relatively little progress has been made towards the development of “next generation” platforms for global protein sequencing at the individual single molecule level.

Furthermore, the relative complexity of protein mixtures such as blood, tissue or cell extracts, as well as the lack of PCR-based amplification or properties such as duplex formation and base-pairing, have hampered the development of single-molecule protein sequencing such as those described for polynucleotides (Harris et aL Science 4 Apr. 2008: Vol. 320. no.

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